This invention relates to a method of treating human immunodeficiency virus (HIV) infection in a mammal comprising administering to the mammal a therapeutically effective amount of a combination of: (i) at least one cytotoxic agent and (ii) at least one non-nucleoside reverse transcriptase HIV inhibitor (NNRTI). This invention also relates to a method of treating chronic viral infections comprising administering to the mammal a therapeutically effective amount of a combination of: (i) at least one cytotoxic agent and (ii) at least one antiviral agent.
HIV infections are at the present time routinely treated with combinations of antiviral drugs (Carpenter, C. et al. Antiretroviral therapy for HIV infection in 1997. JAMA 277, 1962, 1997). The results are beneficial for many patients, with a pronounced drop in viral load, increase in lymphocyte populations, reduction in opportunistic infections and a doubling of time of progression to serious illness and/or death. However, the treatments are done chronically, and can lose effectiveness over time, in part because few if any individuals are cured. Usually HIV persists in cells in the immune system as well as in nervous tissue, and the residual virus may, after months or years of persistence and low-level replication, develop resistance to the drugs and reemerge. At that time, a new set of antivirals is substituted for the initial therapy, and the virus may again be suppressed, although frequently less effectively. The decline in circulating, functional lymphocytes is not only characteristic of HIV infection, it is believed to play a central role in the loss of immunity in the patients (Meyaard, L. et al. Programmed death of T-cells in HIV-1 infection. Science 257, 217, 1992; Ameisen, J. et al. Relevance of apoptosis to AIDS pathogenesis. Trends Cell Biol. 5, 27, 1995). For example, CD4+lymphocytes, in which the HIV preferentially replicates, may decline by 90-99% in the final stage of the HIV infection. The current measure of success of conventional HIV antiviral therapy is to protect these cells and restore their numbers and functions. HIV-infected cells are known to be more susceptible to the toxic effects of cytocidal drugs, certain cytokine proteins, radiation and other damaging agents, compared to their uninfected counterparts (Wong, G. et al. TNF alpha selectively sensitizes HIV-infected cells to heat and radiation. PNAS 88, 4372, 1991; Sandstrom, P. et al. HIV gene expression enhances T-cell susceptibility to H2O2-induced apoptosis. AIDS Res. and Human Retro. 9, 1107, 1993; Katsikis, P. et al. Fas antigen stimulation induces marked apoptosis of T-lymphocytes in HIV-infected individuals. J. Exp. Med. 181, 2029, 1995; Wu X. et al. Apoptosis of HIV-infected cells following treatment with Sho-saiko To and its components. Jpn. J. Med. Sci. Biol. 48, 79, 1995; Gougeon, M. et al. Programmed cell death in peripheral lymphocytes from HIV-infected persons. J. Immunol. 156, 3509, 1996; Hashimoto, K. et al. Stavudine selectively induces apoptosis in HIV type 1-infected cells. AIDS Res. and Human Retro. 13, 193, 1997; Prati, E. et al. Study of spontaneous apoptosis in HIV and patients: Correlation with clinical progression and T-cell loss. AIDS Res. and Human Retro. 13, 1501, 1997). The explanation of the events leading to enhanced death of HIV-infected cells is controversial, but it is clear that the infected cells become depleted of certain broadly acting anti-death proteins normally present in the cells, including the well-known bcl-2 family of proteins (Reed, J. Bcl-2 and the regulation of programmed cell death. J. Cell Biol. 124, 1, 1994). A decrease of bcl-2 typically leads to onset of programmed cell death, or apoptosis, a phenomenon widely observed in HIV-infected cells in vivo and in vitro, both spontaneously and following various external stresses (Liles, W. C. Apoptosis-role in infection and inflammation. Curr. Opin. Inf. Dis. 10, 165, 1997).
The tendency of HIV-infected cells to die or be selectively killed by toxic treatments is known. However, prior to this invention this knowledge has not been exploited for therapeutic purposes. The reason for this is that virtually all cytotoxic treatments cause rapid activation of HIV transcription, leading to a large burst of new progeny virus which proceeds to infect surrounding healthy cells and spread the infection. There has been speculation that the unusually high level of cell killing (for a retrovirus) is part of the biology of HIV, leading to greater spread of the infection (Matsuyama, T. et al. Cytocidal effect of TNF on cells chronically infected with HIV: Enhancement of HIV replication. J. Virol. 63, 2504, 1989; Woloschak, G. et al. HIV expression in dying cells. Bioch. Bphys. Acta 1351, 105, 1997; Zhang, Y. et al. Induction of apoptosis by primary HIV-1 isolates correlates with productive infection in peripheral blood mononuclear cells. AIDS 11, 1219, 1997). Standard antiviral drugs, such as azidothymidine, are not powerful enough to keep HIV in check if infected cells are exposed to a potent cytotoxic drug. This has severely complicated efforts to use chemotherapy to control cancers in HIV-infected individuals (Levine, A. et al. Low-dose chemotherapy with CNS prophylaxis and Zidovudine maintenance in AIDS-related lymphoma. JAMA 266, 84, 1991; Zanussi, S. et al. Effects of anti-neoplastic chemotherapy on HIV disease. AIDS Res. and Human Retro. 12, 1703, 1996).
A number of new antiviral agents have been developed, such as efavirenz, which potently inhibit the reverse transcriptase of the virus. We have discovered in studies in cell culture that such a potent reverse transcriptase inhibitor, combined with a standard cytotoxic drug such as etoposide, can inhibit HIV replication, while infected cells are selectively killed. Surprisingly, a different class of anti-HIV agents, namely protease inhibitors, failed to permit eradication of infected cells.
One object of the present invention is to provide a novel method of treating human immunodeficiency virus (HIV) infection in a mammal comprising administering to the mammal a therapeutically effective amount of a combination of: (i) at least one cytotoxic agent, and (ii) at least one non-nucleoside reverse transcriptase HIV inhibitor (NNRTI).
Another object of the present invention is to provide a novel method of treating chronic viral infections including, but not limited to, those caused by herpesvirus, cytomegalovirus, hepatitis B virus, hepatitis C virus, and varicella-zoster by selectively eradicating the virally infected cells, comprising administering to the mammal a therapeutically effective amount of: (i) at least one cytotoxic agent, and (ii) and at least one antiviral agent selective for the chronic virus.
These and other objects, which will become apparent during the following detailed description, have been achieved by the inventor""s discovery that the administration of a cytotoxic agent, component (i) in combination with a NNRTI, component (ii) results in an unexpected, selective eradication of HIV infected cells.
Preferred embodiments of the invention have been chosen for purposes of illustration and description, but are not intended in any way to restrict the scope of the invention.
In a first embodiment, the present invention provides a method of treating human immunodeficiency virus (HIV) infection in a mammal comprising administering in combination to the mammal a therapeutically effective amount of: (i) a cytotoxic agent and (ii) at least one non-nucleoside reverse transcriptase HIV inhibitor (NNRTI).
In a preferred embodiment, the NNRTI is efavirenz.
In another preferred embodiment, the cytotoxic agent is losoxantrone.
In a more preferred embodiment, cytotoxic agent is losoxantrone, and the NNRTI is efavirenz.
In another embodiment, the present invention provides a method of treating chronic viral infections including, but not limited to, those caused by herpesvirus types I and II, cytomegalovirus, hepatitis B virus, hepatitis C virus, and varicella-zoster by selectively eradicating the virally infected cells, comprising administering to the mammal a therapeutically effective amount of: (i) at least one cytotoxic agent, and (ii) and at least one antiviral agent selective for the chronic virus.
The cytotoxic compound losoxantrone is described in U.S. Pat. No. 4,556,654, 4,608,439, and 4,672,129, such disclosures are hereby incorporated by reference.
The non-nucleoside reverse transcriptase inhibitor of HIV, efavirenz, is described in U.S. Pat. No. 5,519,021, 5,663,169, and 5,665,720, such disclosures are hereby incorporated by reference.
As used herein, the term xe2x80x9cnon-nucleoside reverse transcriptase HIV inhibitorxe2x80x9d (NNRTI) includes, but is not limited to, delavirdine, (Pharmacia and Upjohn U90152S), efavirenz (DuPont Pharmaceuticals), nevirapine (Boehringer Ingelheim), RO 18,893 (Roche), trovirdine (Lilly), MKC-442 (Triangle), HBY 097 (Hoechst), ACT (Korean Research Institute), UC-781, (Rega Institute), UC-782, (Rega Institute), RD4-2025 (Tosoh Co. Ltd. ), MEN 10970 (Menarini Farmacuetici), TIBO derivatives, BI-RG-587, L 697,661, LY 73497, and loviride (Jannsen). additional examples include (xe2x88x92)-6-chloro-4-cyclopropylethynyl-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinone), xe2x80x9cCompound Axe2x80x9d; (+)-4-Cyclopropylethynyl-5,6-difluoro-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinone), xe2x80x9cCompound Bxe2x80x9d; (+)-4-cyclopropylethenyl-5,6-difluro-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinone, xe2x80x9cCompound Cxe2x80x9d; and (xe2x88x92)-6-chloro-4-E-cyclopropylethenyl-4-trifluoromethyl-3,4-dihydro-2(1H)-quinazolinone, xe2x80x9cCompound Dxe2x80x9d (DuPont Pharmaceuticals), NNRTIs disclosed in U.S. application Ser. No. 09/056820, the disclosure of which is hereby incorporated by reference.
As used herein, the term xe2x80x9ccytotoxic agentxe2x80x9d includes, but is not limited to, altretamine, busulfan, chlorambucil, cyclophosphamide, ifosfamide, mechlorethamine, melphalan, thiotepa, cladribine, fluorouracil, floxuridine, gemcitabine, thioguanine, pentostatin, methotrexate, 6-mercaptopurine, cytarabine, carmustine, lomustine, streptozotocin, carboplatin, cisplatin, oxaliplatin, iproplatin, tetraplatin, lobaplatin, JM216, JM335, fludarabine, aminoglutethimide, flutamide, goserelin, leuprolide, megestrol acetate, cyproterone acetate, tamoxifen, anastrozole, bicalutamide, dexamethasone, diethylstilbestrol, prednisone, bleomycin, dactinomycin, daunorubicin, doxirubicin, idarubicin, mitoxantrone, losoxantrone, mitomycin-c, plicamycin, paclitaxel, docetaxel, topotecan, irinotecan, 9-amino camptothecan, 9-nitro camptothecan, GS-211, etoposide, teniposide, vinblastine, vincristine, vinorelbine, procarbazine, asparaginase, pegaspargase, octreotide, estramustine, hydroxyurea.
xe2x80x9cTherapeutically effective amountxe2x80x9d is intended to include an amount of a compound or an amount of a combination of compounds claimed effective to inhibit HIV infection or treat the symptoms of HIV infection in a host. The combination of compounds is preferably a synergistic combination. Synergy, as described for example by Chou and Talalay, Adv. Enzyme Regul. 22:27-55 (1984), occurs when the effect (in this case, eradication of HIV-infected cells) of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is most clearly demonstrated at suboptimal concentrations of the compounds. Synergy can be in terms of lower cytotoxicity, increased antiviral effect, or some other non-additive beneficial effect of the combination compared with the individual components.
By xe2x80x9cadministered in combinationxe2x80x9d, xe2x80x9ccombinationxe2x80x9d, or xe2x80x9ccombinedxe2x80x9d when referring to component (i) and component (ii) of the present invention, it is meant that the components are administered concurrently to the cell or mammal being treated. When administered in combination each component may be administered at the same time or sequentially in any order or at different points in time. Thus, component (i) and component (ii) may be administered separately but sufficiently closely in time so as to provide the desired HIV-infected cell eradication effect.
As used herein, xe2x80x9cselectivexe2x80x9d refers to the ability of a particular drug or antiviral protein to target a cell that is infected with a specific virus.
As used herein, xe2x80x9cantiviral agentxe2x80x9d includes, but is not limited to, lamivudine (3TC), famcyclovir, lobucavir, adevovir, interferon alpha, interferon alpha plus virazole, acylovir, valacyclovir, sorivudine, iododeoxyuridine, gancyclovir, foscavir, cidofovir, fomivirsen, and netivudine.
Pharmaceutical kits useful for the inhibition of HIV and treatment of HIV infection, which comprise a therapeutically effective amount of a pharmaceutical composition comprising a compound of component (i) and one or more compounds of component (ii), in one or more sterile containers, are also within the ambit of the present invention. Sterilization of the container may be carried out using conventional sterilization methodology well known to those skilled in the art. Component (i) and component (ii) may be in the same sterile container or in separate sterile containers. The sterile containers of materials may comprise separate containers, or one or more multi-part containers, as desired. Component (i) and component (ii), may be separate, or physically combined into a single dosage form or unit as described above. Such kits may further include, if desired, one or more of various conventional pharmaceutical kit components, such as for example, one or more pharmaceutically acceptable carriers, additional vials for mixing the components, etc., as will be readily apparent to those skilled in the art. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and/or guidelines for mixing the components, may also be included in the kit.
In order to test whether some external death stimuli, which are independent of bcl-2 (Reed, 1994), may not selectively kill HIV-infected cells, we exposed uninfected and HIV-infected cells to various chemical toxins, including losoxantrone, bisnafide, etoposide and hydroxyurea. Etoposide is the only one of these agents to have previously been shown to be blocked by bcl-2 (Reed, 1994). In cultured cells expressing an increased level of bcl-2, we found a 3 to 5-fold resistance to the actions of etoposide and losoxantrone, but not bisnafide. Therefore, we proceeded to examine whether either losoxantrone or etoposide were able to selectively kill HIV-infected cells. Table 1 shows the results of such an experiment.
After 24 hours in presence of the cytotoxic chemicals, losoxantrone had killed infected cells to a 5-fold greater extent than uninfected lymphocytes, while etoposide was 6-fold more toxic to infected cells. Efavirenz showed no toxicity to either cell population at the concentration shown, 10 nM.
Comparing cell survival for infected vs. control cells indicates there is a range of losoxantrone concentrations that show selectivity for infected cells from 0.1 to 10 nM. At 10 nM or greater, there is evidence of substantial toxicity to uninfected cells, but selectivity is still not entirely lost.
Previous studies from several laboratories indicated that although selective killing of HIV-infected cells follows a variety of toxic treatments, it was always accompanied by a large burst of HIV transcription and release of progeny virus particles, which could then proceed to infect and replicate in neighboring cells (Wong, 1991; Matsuyama, 1989; Woloschak, 1997; Zhang, 1997; Levine, 1991; and Zanussi, 1996). This phenomenon has made dealing with solid tumors or leukemias/lymphomas very difficult in HIV-positive individuals (Levine, 1991; and Zanussi, 1996), in part because early HIV inhibitors which could be co-administered had relatively low potency to inhibit the virus replication and spread, particularly after a strong induction by a cytotoxic drug. Recently, the situation has changed, with introduction of potent antivirals directed at the HIV protease or reverse transcriptase (Carpenter, 1997).
In order to selectively kill infected cells without an accompanying induction of HIV, we combined a very potent antiviral with a cytotoxic drug. The experimental protocol we adopted to monitor eradication of infected cells is based on the principle that surviving infected cells will produce sufficient HIV to initiate a new wave of infection in naive cells, in a co-cultivated environment. This is sometimes called an infectious center assay procedure, and has been used for HIV and other viruses (The Molecular Biology of Poliovirus. F. Koch and G. Koch, eds. p307. Springer Verlag, Vienna 1985).
Minimal doses of the compounds were employed in our trial. For the cytotoxic drugs losoxantrone and etoposide, a dose was selected which would be toxic to 90% or greater of HIV-infected cells, but non-toxic to uninfected cells. For the anti-retrovirals, namely the reverse transcriptase inhibitor efavirenz or the cyclic urea HIV protease inhibitor [4R-(4xcex1, 5xcex1, 6xcex2, 7xcex2]-1,3-bis (3-aminophenylmethyl]hexahydro-5,6-dihydroxy-4,7-bis (phenlymethyl-2H-1,3-diazepin-2-one dimethylsulfonate (xe2x80x9cCompound Exe2x80x9d, DuPont/Triangle), the dose chosen was approximately the IC90 for HIV replication. This minimal dose was used so that further losses of HIV infectivity due to application of the cytotoxic drug could easily be monitored; in a clinical setting the highest tolerated antiviral dose would be preferred.
Freshly isolated human blood lymphocytes and monocytes were infected with HIV-1 (RF strain) and then incubated for an additional 3 days in the presence of compounds alone or in combinations or left untreated. At the end of 4 days, the infected cells were briefly centrifuged. The supernatant was collected and used to titer HIV p24, using an enzyme-linked immunosorbent assay (ELISA). The cell pellets were washed two times with medium lacking any inhibitors, and the washed human peripheral blood mononuclear cells (PBMCs) were mixed 1:1 with MT-2 lymphocytes and incubated for an additional 4 days absent any inhibitors. All cells were then removed by centrifugation and the supernatant was again assayed for p24 levels by ELISA.
As shown in Table 2 below, losoxantrone, efavirenz and Compound E, at the concentrations shown, caused only small changes in the level of HIV p24 after either 4 or 8 days.
Losoxantrone(10 nM) caused a 3 to 7-fold increase in p24 titers, while the two antivirals caused about a 80% reduction on day 4, which had recovered by day 8, after compounds were removed. Only the combination of a cytotoxic drug with efavirenz caused a substantial loss (1 log or greater) both at days 4 and 8. It was noted that even after removal of both compounds and 4 further days of incubation, there was little or no p24 production by the MT-2 cells, including a large loss of infectious centers among the PBMCs. In some experiments, there was no p24 detected above background at day 8 in the cells treated with efavirenz and losoxantrone. When etoposide was substituted for losoxantrone, the results were very similar (not shown). In comparison, there was little effect on p24 production by combining the protease inhibitor, Compound E with a cytotoxic drug.
Recent reports have indicated a synergy between a reverse transcriptase inhibitor, didanosine (DDI) and a cytotoxic agent, hydroxyurea (HU) (Lori, F. et al. Hydroxyurea as an inhibitor of HIV-1 replication. Science 266, 801, 1994; Lori, F. et al. Combination of a drug targeting the cell with a drug targeting the virus controls HIV-1 resistance. AIDS Res. and Human Retro. 13, 1403, 1997). We therefore tested whether HU could be combined with efavirenz to eradicate infectious centers. A dose of HU was chosen which was shown to inhibit PBMC cell division by 3-fold (100 xcexcM). However, the combination of efavirenz with that dose of HU failed to significantly reduce the number of infectious centers as shown in Table 3.
Leveraging the tendency of HIV-infected cells to die or be killed has been problematic because of the coincident burst of new HIV progeny upon exposure to cytocidal drugs, toxic cytokines or physical treatments (Wong, 1991; Matsuyma, 1989; Woloschak, 1997; Zhang, 1997; Levine, 1991; and Zanussi, 1996). First-generation antiretroviral agents were not powerful enough to hold the virus in check under such conditions, and the currently available protease inhibitors fail to permit the selective killing action by tumoricidals such as losoxantrone or etoposide (Table 2). However, the use of the potent reverse transcriptase inhibitor efavirenz allows coincident application of a cytotoxic drug, with subsequent loss of infectious centers and suppression of virus production (Table 2). The combined use of the two agents is thus able to effect what no combination of antivirals has so far: the selective elimination of infected cells. In the clinic, a non-cytotoxic dose (for uninfected cells, which constitute the majority of cells even in a patient with full-blown AIDS) of a cytocidal drug should lead to selective killing of infected cells, and coupled with potent inhibition of the virus by efavirenz, little or no spread of infection should occur.
A significant advantage of this invention""s approach, which combines a cytotoxic agent with one or more non-protease inhibitor antiretrovirals, is expected to be a rapid improvement in patient health, with no need for repeated, long-term dosing. Infected cells and virus should be quickly eliminated and this could significantly reduce the cost of treatment, and make therapy much more widely available in developing countries. Also, the acute nature of our therapeutic approach should minimize the ability of HIV to develop resistance, which may occur with conventional treatment regimens (Carpenter, 1997).